This application is designed to identify compounds to treat Charcot-Marie-Tooth type 2E (CMT2E). CMT is the most commonly inherited neurological disorder with a reported prevalence of 1 in 2,500 people worldwide. It is found in all races and ethnic groups. CMT is slowly progressive and CMT patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. The nerve degeneration causes the subsequent degeneration of the muscles in the extremities. Among the symptoms of CMT are foot-drop, steppage gait, high arches, foot bone abnormalities, and basic problems with hand function, as well as sometimes breathing difficulties. CMT is not usually life threatening, but can cause severe disabilities. Although the genes mutated in CMT are also expressed in the central nervous system, the disorder almost never affects brain function. CMT is divided in two major types, CMT1 and CMT2, based on nerve conduction velocities, which are reduced in CMT1 and relatively normal in CMT2. In general, CMT1 is a demyelinating neuropathy caused by mutations in genes important in myelin formation, whereas CMT2 is axonal. Mutations in the neuronal intermediate filament gene, NEFL have been shown to cause a subtype of CMT2, called CMT2E. NEFL encodes the neurofilament light (NFL) protein that we have shown to be a necessary component for the assembly of neuronal intermediate filaments. Mutations in this gene are responsible for approximately 2% of all CMT cases. Neuronal intermediate filaments form the intermediate filament network in neurons and are the predominant cytoskeletal structure in the axon. Neurofilamentous aggregates both in the neuronal cell bodies and axons are seen in patients with mutations in NEFL, as well as in other neurodegenerative diseases, such as amyotrophic lateral sclerosis. We have shown that the mutant NFL proteins form aggregates in transfected neuronal and non-neuronal cells. Misassembled neurofilament aggregates are found in all transfected cells expressing the pathogenic CMT-associated NFL mutant proteins, but not in cells expressing several polymorphic variants that are non-pathogenic. We hypothesize that inhibitors of neurofilament misassembly will lead to therapies for CMT. Therefore, the goals of this proposal are to identify small molecules that inhibit misassembly and to test their effects in a mouse model of CMT2E. We will focus our attention on two of the first described mutant NFL proteins, P8R NFL and Q333P NFL that we have characterized in the most detail. Using high-throughput visual screening methodology, we will identify small molecules that inhibit neurofilament misassembly. For the second and related part of the project, we will generate knock-in mouse models of CMT2E with mutations in the Nefl gene. These mice will be characterized phenotypically and we will then test compounds identified in the visual screens for their ability to ameliorate peripheral neuropathy in these mouse models. The proposed research will identify lead compounds for the development of drugs to treat human subjects with CMT, as well as potentially other neurodegenerative diseases and it will also generate mouse models of a human hereditary neuropathy that will be of value to many other investigators in the field. PUBLIC HEALTH RELEVANCE: CMT is the most commonly inherited form of peripheral neuropathy. The goal of this project is to use chemical screening to identify novel drugs to treat one type of CMT and test them in mouse models of the disease.